Our group has determined that PIAS3-dependent SUMOylation plays a central role in regulating both rod and cone photoreceptor differentiation. However, several critical questions about the function of PIAS3 and protein SUMOylation in photoreceptors still remain unresolved. While protein SUMOylation is essential for photoreceptor viability, our previous work did not determine whether PIAS3 is required for the maturation, maintenance or survival of differentiated photoreceptors. To address this question, we will generate and characterize mutant mice that selectively lack PIAS3 expression in photoreceptors. Second, it is not known how PIAS3-dependent SUMOylation induces NR2E3 to act as a transcriptional repressor when bound to the promoters of cone-specific genes, but does not do so when it bound to the promoters of rod-specific genes. We will test whether the TFIIH-associated proteins ERCC2 and CDK7 might mediate this effect by blocking PIAS3-dependent SUMOylation of NR2E3 when it is bound to the promoters of rod-specific genes. Finally, preliminary studies of SUMO ligase specificity obtained using protein microarrays have suggested that PIAS3 and TOPORS, another E3 SUMO ligase critical for photoreceptor function, may regulate the activity of multiple photoreceptor-specific proteins that function outside the nucleus. We have found that BBS4 is SUMOylated by PIAS3, and propose to study how this process regulates BBSome assembly and rhodopsin transport in vivo. We also plan to investigate whether PIAS3 also regulates the function of other ciliary and centrosomal proteins such as CEP290 through site-specific SUMOylation, and will whether the activity of PIAS3 is controlled by TOPORS-dependent SUMOylation, as suggested by our preliminary in vitro analysis.